Protein protease inhibitors of the serpin superfamily play important roles in regulating intracellular and extracellular serine and cysteine proteases in numerous physiologic processes. Serpins regulate proteolytic enzymes through a novel inhibition mechanism in which the protease is trapped at the acyl-intermediate stage of proteolysis of the serpin as a regular substrate due to major conformational changes induced in both the serpin and protease. The long term goal of the proposed studies is to answer outstanding questions concerning this unusual conformational trapping mechanism and how various accessory ligands modulate this mechanism. The knowledge gained from these studies is expected to enhance understanding of the complex modes by which serpins regulate proteolysis and provide new insights into how natural serpin mutations disrupt this regulation. The proposed studies will test the hypotheses that i) serpins trap proteases of different structural families in stable acyl-intermediate complexes by different mechanisms; ii) the serpin F helix plays an essential active role in the serpin inhibitory mechanism and iii) the protein Z-dependent serpin, ZPI, regulates factor Xa in procoagulant complexes by binding protein Z and recognizing membrane-bound factor Xa through exosite interactions residing in protein Z and ZPI. These hypotheses will be tested by the following specific aims: 1) we will assess the relative importance of i) disrupting serpin reactive loop-protease interactions, ii) distorting the protease and iii) binding of the distorted protease to the serpin in stabilizing serpin-protease acyl-intermediate complexes for proteases of different mechanistic class, structure and specificity; 2) we will elucidate whether the F helix plays an active role in coupling the energy of the serpin reactive loop conformational change to distorting the protease in the acyl-intermediate complex through reversible movements or structural changes in the helix; and 3) we will determine the structural requirements which mediate serpin specificity and the regulation of serpin function in the factor Xa-specific serpin, ZPI. The proposed studies will utilize mutagenesis, fluorescence, NMR and X-ray crystallography and thermodynamic and kinetic approaches to characterize serpin-protease and serpin-ligand interactions.